Bacteria are exposed to a variety of environmental conditions and have evolved different mechanisms to cope with changes in the extracellular milieu. The existence of bacterial K+/H+ antiporters that prevent the over- accumulation of potassium in the cytoplasm was predicted by Peter Mitchell over 50 years ago and K+/H+ antiport activity as such was demonstrated in everted membrane vesicles from Escherichia coli some time ago. However, despite the importance of K+/H+ antiport for bacterial physiology, its molecular mechanisms remain under-investigated and identification of specific housekeeping K+/H+ antiporters in bacteria remained elusive until recently, when we showed that the V. cholerae NhaP2 antiporter acts as a specific K+/H+ antiporter. This protein was most active at low pH and is indispensable for the growth of V. cholerae under those conditions, removing excess of internal K+ from the cytoplasm at the expense of pH. We have also begun to characterize the other two NhaP-type antiporters from V. cholerae. Here, we propose to further examine the physiological roles of the different NhaP paralogs in Vibrio cholerae. Like most pathogens, Yersinia pestis have to adapt to wide range of environments to establish an infection. One of the most peculiar features of the Y. pestis physiology and virulence is its stringent dependence on alkali/alkaline cations, primarily Ca2+. Despite the importance of calcium in the regulation of Y. pestis virulence factor production, virtually nothing is known abou the mechanism of membrane transport of calcium in this organism. In Y. pestis, several different proteins potentially mediate the transport of Ca2+ across the bacterial membrane and some of these proteins are predicted to be able to integrate Ca2+, Na+, K+ and pH homeostasis in this organism. The present project is focused on the importance of the various putative calcium transporters encoded by Y. pestis for bacterial growth under various conditions. As the function and expression of the T3SS appears to be sensitive to both Ca2+ and (indirectly) Na+, we will also examine Yop secretion in these mutants to allow the assessment of their potential as targets for the development of a novel class of antimicrobials. Our experimental approach is expected to yield the following outcomes. It is anticipated that, at the end of the requested two years of support, we will have analyzed the kinetics and cation selectivity of various mutant NhaPs, thereby testing the validity of our ligand shading hypothesis. In addition, we will have identified the detailed roles of the three NhaP paralogs in the overall membrane bioenergetics and physiology of V. cholerae. Lastly, we expect to have gained important information about the link between membrane calcium transport and virulence of Y. pestis. Thus, our proposed work has the potential to add significantly to our understanding of the contribution of membrane bioenergetics to bacterial physiology per se. Moreover, our work will begin to evaluate the role of NhaP-mediated cation transport in bacterial pathogenesis that could provide the basis for future testing of nhaP mutant strains in animal models. Ultimately, we anticipate the identification of transporters that might provide novel targets for drug development during later phases of the project, potentially resulting in the development of a new class of effective anti-bacterial agents with completely new mechanisms of action.